An improved synthesis of poly(amidoamine)s for complexation with self-amplifying RNA and effective transfection
An improved synthesis of poly(amidoamine)s for complexation with self-amplifying RNA and effective transfection
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DOI：
10.1039/d0py00912a
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发表时间：
2020-09-28
影响因子：
4.6
通讯作者：
Alexander, Cameron
中科院分区：
文献类型：
--
作者：
Gurnani, Pratik;Blakney, Anna K.;Alexander, Cameron
阳离子聚合物被广泛用作用于基于基因的治疗的核酸缩合材料。这些疫苗的开发主要是为了提供用于癌症治疗的DNA和RNA，但正在进行的COVID-19大流行表明迫切需要新的DNA和RNA疫苗。考虑到这一点，需要用于这种阳离子聚合物的合适的制造条件，其可以在配制和递送阶段保护核酸，但在正确的细胞区室中有效且安全地释放货物。许多基于聚（酰胺基胺）的聚合物符合这些标准，但它们的合成可能是耗时的、低效的和可再现性差的，排除了它们作为可制造的疫苗赋形剂的采用。本文报道了一种改进的聚（胱胺双丙烯酰胺-co-4-氨基-1-丁醇）（简称pABOL）的合成方法。单体含量和化学计量、溶剂、稀释剂和温度的优化，结合微波的应用，使得能够在4小时内制备疫苗候选pABOL材料，而先前的合成报道为48小时。这些程序在多次重复合成中具有高度重现性。用模型RNA的转染实验显示，具有适当摩尔质量和质量分布的制剂聚合物在模型细胞系中与衍生自未优化合成的聚合物一样有效，所述聚合物已显示出作为RNA疫苗制剂候选物具有高功效。
Cationic polymers are widely used as materials to condense nucleic acids for gene-based therapies. These have been developed to mainly deliver DNA and RNA for cancer therapies but the ongoing COVID-19 pandemic has demonstrated an urgent need for new DNA and RNA vaccines. Given this, suitable manufacturing conditions for such cationic polymers which can protect the nucleic acid in the formulation and delivery stages but release the cargo in the correct cellular compartment effectively and safely are required. A number of polymers based on poly(amidoamine)s fit these criteria but their syntheses can be time-consuming, inefficient and poorly reproducible, precluding their adoption as manufacturable vaccine excipients. Here we report an improved synthesis of poly(cystamine bisacrylamide-co-4-amino-1-butanol), abbreviated as pABOL,viamodifications in concentration, reaction time and reaction conditions. Optimisation of monomer contents and stoichiometries, solvents, diluents and temperature, combined with the application of microwaves, enabled the preparation of vaccine candidate pABOL materials in 4 h compared to 48 h reported for previous syntheses. These procedures were highly reproducible in multiple repeat syntheses. Transfection experiments with a model RNA showed that polymers of formulation with appropriate molar masses and mass distributions were as effective in model cell lines as polymers derived from the unoptimised syntheses which have been shown to have high efficacy as RNA vaccine formulation candidates.